Isotopically modified compounds and their use as food supplements

ABSTRACT

A nutrient composition comprises an essential nutrient in which at least one exchangeable H atom is  2 H and/or at least one C atom is  13 C. The nutrient is thus protected from, inter alia, active oxygen species.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/446,922, filed Mar. 1, 2017, which is a divisional application ofU.S. patent application Ser. No. 15/078,853, filed Mar. 23, 2016 andissued as U.S. Pat. No. 9,616,042, which is a divisional application ofU.S. patent application Ser. No. 14/551,450, filed Nov. 24, 2014 andissued as U.S. Pat. No. 9,320,289, which is a divisional application ofU.S. patent application Ser. No. 12/281,957, filed Aug. 17, 2009 andissued as U.S. Pat. No. 8,906,405, which claims priority to U.S.National Phase under 35 U.S.C. § 371 of International Application No.PCT/GB2007/050112, filed Mar. 8, 2007, which claims priority to UnitedKingdom Application No. 0604647.8, Filed Mar. 8, 2006. The disclosuresof the above-referenced applications are hereby expressly incorporatedby reference in their entireties.

FIELD OF THE INVENTION

The present Invention related to isotopically modified compounds andtheir use as food supplements.

BACKGROUND OF THE INVENTION

A currently accepted theory of ageing blames the irreversible changes incell machinery and reduced efficiency of metabolic processes on thedetrimental effects of free radicals and other reactive oxygen species(ROS) or reactive nitrogen species (RNS) which are normally present inthe cell as part of the respiratory process. ROS and RNS oxidize/nitrateDNA, proteins, lipids and other cell components. Of these, proteinoxidation, which converts arginine, lysine, threonine, thryptophan andproline into corresponding carbonyl compounds, cannot be repaired byproteases after a certain threshold number of amino acid residues havebeen oxidized.

The damaged protein loses its catalytic or structural activity, butproteases are unable to disintegrate heavily carbonylised strands, sothat the damaged species accumulate and aggregate, clogging up cellularpassages. This rust-like process gradually wears down all cellularmechanisms, slowing everything down and ultimately causing cellulardeath.

Apart from ageing, many diseases such as Alzheimer's, Parkinson's,dementia, cataract, arthritis, chronic renal failure, acute respiratorysyndrome, cystic fibrosis, diabetes, psoriasis and sepsis, to give a fewexamples, are associated with increased protein carbonylation.Typically, physiological levels of protein carbonyls are at around 1nmol/mg protein, whereas pathological levels go to 8 nmol/mg and above.

For the two molecules involved in the process of oxidative damage ofproteins, i.e. an oxidizer and its substrate, the oxidizer has been thesubject of many studies aiming at neutralizing or removing it by meansof increasing the number of antioxidants (vitamins, glutathione,peptides or enzymes). The substrate, e.g. amino acid (AA) residues whichare converted into carbonyls, has received less attention.

One common feature of all the AA residues (except proline) vulnerable tocarbonylation is that they belong to the group of essential AAs, whichcannot be synthesized by vertebrata and should be ingested, e.g.consumed with food. The group includes phenylalanine, valine,tryptophan, threonine, isoleucine, methionine, histidine, arginine,lysine and leucine (arginine is essential for children of up to 5 yearsof age).

Oxidation of both Arg and Lys by ROS yields aminoadipic semialdehyde andproceeds through sequential replacement of co-hydrogens with hydroxyls.Oxidation of Lys, Arg, Tip, Thr, Phe and His is shown in FIG. 1.Side-chains undergo the same transformations if these AAs are part ofpolypeptides/proteins. Other essential AAs undergoing ROS-drivenoxidation include Leu (to 5-hydroxyleucine), Val (3-hydroxy valine) andIle (several products).

Other types of oxidative damages affecting essential AAs involvereactive nitrogen species (RNS). Examples are shown in FIG. 2.

Yet another process detrimental to proteins is a ROS-driven peptide bondcleavage, which is preceded by oxygen free radical-mediated proteinoxidation. A hydrogen atom is abstracted from a Ca atom of thepolypeptide chain, which then leads to formation of an alkoxyl radical.This can lead either to hydroxyl protein derivative, or to peptide bondcleavage by (1) diamide or (2) α-amidation pathway. This is illustratedis FIG. 3.

Nucleic acids are not normally considered as essential components of thediet, but are also damaged by ROS. An example particularly important forthe mitochondrial functioning is the formation of 8-oxy-G, asillustrated in FIG. 4. This leads to mutations in the mitochondrialgenome, which is not maintained and repaired as efficiently as thenuclear genome, with detrimental consequences to the efficiency ofrespiratory processes in the cell. Another cause of degradation isradiation.

The kinetic isotope effect is widely used when elucidating mechanismsand rate-determining stages of chemical and biochemical reactions. Therate of reaction involving C—¹H bond cleavage is typically 5 to 10 timesfaster than the corresponding C—²H (²H−D=deuterium) bond cleavage, dueto the two-fold difference in the masses of H and D isotopes. Thedifference in reaction rates is even higher for tritium (³H or T) as itis 3 times heavier than hydrogen, but that isotope is unstable. Thesecond component of the C—H bond, the carbon atom, can also besubstituted for a heavier ¹³C isotope, but the bond cleavage ratedecrease will be much smaller, since ¹³C is only a fraction heavier than¹²C. See Park et al, JACS (2006) 128: 1868-72.

Oxidation reactions are a good example of the isotope effect, as thehydrogen subtraction by an oxidizer is usually a rate-limiting step ofthe process. Damgaard, Biochemistry (1981) 20: 5662-69, illustratesthis: the kinetic isotope effect upon VZK for (1-R)[1-²H₂]— and(1-R)[I—³H₂]— ethanol oxidation by liver alcohol dehydrogenase (ADH) toacetaldehyde, measured at pH 6, was 3 (D(V/K)) and 6.5 (T(V/K)),decreasing to 1.5 and 2.5 respectively at pH 9. Lower than expectedrates confirm the discrete role of the non-ADH systems as alternativepathways. In vivo experiments in perfused rat liver, as reported inLundquist et al, Pharm, & Tox. (1989) 65: 55-62, gave the mean value ofD(V/K) of 2.89. Therefore, in all cases the oxidation of deuteratedethanol was substantially slowed down.

Isotopically labelled material has been administered to animals, andalso to humans, for diagnostic purposes. Gregg et al, Life Sciences(1973) 13: 755-82, discloses the administration to weanling mice of adiet in which the digestible carbon fraction contained 80 atom % ¹³C.The additive was ¹³C-labelled acetic acid. Tissue examination revealedno abnormalities clearly attributable to the high isotopic enrichment.

SUMMARY OF THE INVENTION

The present invention is based on the realisation that isotopicsubstitution can be used to synthesize a class of compounds that, wheningested, result in the formation of bodily constituents (e.g. proteins,nucleic acids, fats, carbohydrates, etc) that are functionallyequivalent to normal bodily constituents but which have a greaterresistance to degradative/detrimental processes, e.g. those mediated byROS and RNS or radiation. Therefore, according to this invention, anutrient composition comprises a nutrient composition comprising anessential nutrient in which at least one exchangeable H atom is ²Hand/or at least one C atom is ³³C.

Compounds for use in the invention are identical to normal nutrients orconstituents of food except that they contain stable isotopes which,when Incorporated into bodily constituents make such bodily constituentsmore resistant to degradative processes than they would be otherwise.They provide a method for protecting the preferred functionality ofnatural biomolecules; the method comprises supply of a compound in sucha way that it becomes incorporated into biomolecules and in so doingconfers properties on the biomolecule that protect against damaging orunwanted chemical changes.

Compounds for use in the invention may be chemically synthesized and,when ingested by an organism, are metabolized in a way that results inthe incorporation of the compound into a functional biomolecule; theincorporation of the compound resulting in the biomolecule having ahigher degree of resistance to damaging molecular changes than would bethe case for the equivalent biomolecule that did not comprise thecompound. Such compounds may act as mimics of naturally occurringprecursor elements of biomolecules. They may mimic an essential aminoacid. The organism is typically a plant, microbe, animal or human.

A compound for use in the invention is typically not degraded by enzymesof the P450 pathway. It can therefore accumulate in a subject for whichit is essential.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 4 each show reactions that degrade essential nutrients.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to the fact that essential supplements mayundergo irreversible chemical transformations such as oxidation,nitration, etc, leading to the onset of senescence or diseases.Essential food components cannot be synthesised de novo by an organism,e.g. mammal, primate or human, and therefore need to be supplied withthe diet. For the purposes of this specification, a nucleic acid isessential, although it may be more properly be described asconditionally essential. Conditionally essential nutrients need to besupplied with the diet under certain circumstances.

For humans, 10 amino acids are essential, i.e. Phe, Val, Trp, Thr, He,Met, His, Leu, Lys and Arg (up to the age of five). Purine andpyrimidine nucleosides are conditionally essential. Essential fattyacids are ω-3 and ω-6, while monounsaturated oleic acid is generallynon-essential.

According to this invention, the proposed undesired effects such asageing/diseases can be slowed down. The compounds consumed should bemodified to slow down the undesired reactions, while still retainingtheir chemical identity. This can be achieved in one embodiment bysubstituting hydrogen atoms subjected to abstraction duringoxidation/oxidative substitution at the most reactive carbon sites, orthe sites known to undergo the ROS/RNS inflicted damage as illustratedon FIGS. 1-4, with deuteriums, which due to the isotope effect slow downthe rate of reactions. Substituting carbons instead of or in addition toH atom substitution may require a greater degree of substitution sinceone does not add so much to the reaction rate decrease (D is twice theweight of H, and ¹³C is less than 10% heavier than ¹²C).

Depending in part of the method of preparation, a compound for use inthe invention may comprise partial or total isotopic substitution. Forexample, deuterium substitution may be only at the one or two hydrogenatoms that are considered chemically exchangable, e.g. at OH or CH₂adjacent to a functional group. Total rather than partial ¹³Csubstitution may often be achieved more effectively.

In a preferred embodiment of the invention, the (or only the)oxidation-sensitive hydrogens should be substituted with deuteriums, tominimize the risk of other metabolic processes slowing down whenfragments of these AAs are used to build up other structures. In specialcases, to further increase the resistance to oxidation, both ¹H and ¹²Cof a H—C bond can be substituted by ²H and ¹³C. To minimize any possiblenegative effect of isotopes, such as unwanted slowing down ofbiochemical reactions that utilise fragments of AAs protected withisotopes, preferably only the most sensitive parts of the AAs should bederivatised, for example, co-atoms of Lys and Arg. Preferred compoundsof this type are

If the oxidative stress is so severe that benefits from protecting thevulnerable sites overweigh potential damaging effects from slowing downother metabolic pathways (as is the case with some diseases), then AAsmore heavily protected with isotopes can be employed, as shown in thefollowing, illustrative formulae

Such derivatives confer protection from the detrimental effectsillustrated in FIGS. 1-4.

As all vertebrata have lost the ability to synthesise the essential AAsand require the outside supply of essential AAs or fatty acids,non-painful ways of delivering the deuterated/deuterated and¹³C-modified AAs into human food sources are possible. For AAs, oneexample of process is to create essential AAs-deficientyeast/algae/bacteria/etc, growing them on appropriate isotopically‘protected’ media/substrates and then feeding the obtained biomass tofish or livestock. The fish or livestock can then be introduced into thefood chain in the normal manner. Another example is by a directpill/supplement-based delivery.

Non-essential components of food are the compounds that can be producedby an organism, such as nucleic acid bases. But when these are consumedas food, some of the non-essential components are digested/used asprecursors for other compounds, but a certain fraction is utilizeddirectly in metabolic processes, e.g. nucleic acid (NA) bases,incorporated into DNA. Therefore, as an example, some of the NA basessupplied with food may be isotopically protected, as shown in thefollowing, illustrative formulae

Such species are less vulnerable to oxidation upon incorporation intoDNA. In other words, the oxidation rate of DNA, including mitochondrialDNA, can be reduced.

Both essential and non-essential components may be administered througha digestive system to achieve a desired effect of slowing downdetrimental changes associated with ageing process and various diseases.Nevertheless, ways other than through the digestive tract, for instanceintravenous delivery, can be envisaged. The important aspect of anydelivery system is to get the isotopically engineered compoundsincorporated into bodily/biochemical constituents.

A composition of the invention can be provided like any food supplement.It typically comprises one or more nutrients in addition to theisotopically labelled essential component. It may comprise plantmaterial, microbial material or animal material. The composition may bea normal foodstuff, a tablet or other solid medicament, or an injectableor other liquid.

The composition may comprise unmodified compounds in addition to thosethat have been labelled. The labelled compound is typically present in alarger amount, and certainly greater than that which may be presentnaturally.

Compounds for use in the invention may be prepared by procedures thatare known or that can be modified as appropriate by one of ordinaryskill in the art, For example, the deuteratcd analogue of Lys,2,6-diaminohexanoic acid-6,6-D₂, may be synthesized from a precursornitrile by hydrogenolysis in D₂ according to standard procedures.

The deuterated analogue of Arg, 2-amino-5-guanidinopentanoicacid-5,5-Z)2, may be synthesized from a corresponding nitrile.

Omithine-D₂, obtained by hydrogenolysis in a way similar to thatdescribed above for Lys, was dissolved in water and mixed with an equalvolume of 0.5M O-methylisourea, pH 10.5, adjusted with NaOH. After 4-5h, 1% TFA was added to stop the reaction. The compound was purified by aRP HPLC (Buffers were A: 0.1% TFA/H₂O; B: 0.1% TFA/(80% MeCN/20% H₂O)),0-65% B over 40 mm. See Kimmel, Methods Enzymol, (1967), 11: 584-589,and Bonetto et al, Anal. Chem. (1997), 69: 13154319.

Cyano-aminoacids are precursors to amino acids. Synthesis ofcyano-aminoacids can be carried out by several routes, starting from avariety of precursors. Alcohols (Davis & Untch, J. Org. Chem. (1981);46: 2985-2987), amines (Mihailovic et al, Tet. Lett. (1965) 461-464),amides (Yamato & Sugasawa, Tet Lett. (1970) 4383-4384) and glycine(Belokon et al, JACS (1985)107: 4252-4259) can all serve as startingmaterials in such syntheses. Some methods can yield both 13C and2H-substituted compounds, while others are only compatible withdeuteration.

Deuteration can be carried out using deuterium gas (for example, asdescribed in White et al, JACS (1994) 116: 1831-1838) or differentdeuterides, for example NaBD₄ (Satoh et al, Tet. Let. (1969) 4555-4558):the choice between these methods should be made based on theavailability and price of the corresponding deuterium derivatives. Someof the strategies tested are described in detail below.

The sites to be protected within essential fatty acids for the purposeof the present invention are the methylene groups of the 1,4-dienesystems (‘bis-allyl’ positions). They are the most reactive, and caneasily be derivatised using a variety of methods. Bromination of thisposition followed by reduction with ²H₂ results in the substitution ofone hydrogen at a time. To substitute both, the procedure should berepeated twice. A more attractive method may be a direct one-stepsubstitution in heavy water. An example of such exchange is given below(Example 6) for 8-deuteration of deoxyguanosine.

An alternative approach to the synthesis of deuterated unsaturated fattyacids is based on strong base treatment of 1,4-dienes followed byquenching with heavy water. This is illustrated in Example 7.

There are literature examples for substitutions at any position for allmajor nucleotide bases, with all major types of isotopes (²H₂— ³H₂, ¹³C,¹⁴C, ¹⁵N, ¹⁸O etc). Described below are just two procedures, based onthe previously published work, for selective deuteration of purines(Esaki et al, Heterocycles (2005) 66: 361-369, and Chiriac et al,Labelled Compd. Radiopharm. (1999) 42: 377-385). Numerous otherprotocols are suitable as well. It is often possible to exchangehydrogens for deuteriums on an existing nucleic acid base/nucleoside,while to incorporate ¹³C, the bases should be assembled (for example,see Folesi, et al, Nucleosides Nucleotides Nucleic Acids (2000).

Syntheses of some isotopically ‘reinforced’ essential dietary componentssuitable for use in the present invention are known; see for instance,6,6-²H₂, 1,1-¹³C₂-L-Lys: Lichtenstein et al, J. Lipid Res. (1990) 31:1693-1701 and 8-deutero-deoxy-guanosine: Toyama et al, J. RamanSpectrosc. (2002) 33: 699-708).

The invention is not limited by the synthetic organic chemistry methodsdescribed above, as there exists a large arsenal of different methodsthat can also be used to prepare the above mentioned and otherisotopically protected components suitable for use in the presentinvention. For instance, in addition to the methods disclosed in theExamples, other methods suitable for convention of a primary amino groupfunction into a CN function (with the aim of subsequent deuteration ofthe alpha-(relative to N) carbon atom) can be employed, such as:

-   -   a direct oxidation by oxygen catalysed by cuprous        chloride-dioxygen-pyridine system (Nicolaou et al,        Synthesis (1986) 453-461: Capdevielle et al, Tet. Lett, (1990)        31: 3305-3308)    -   a direct conversion using bromosuccinimide (Gottardi Monatsh.        Chem. (1973) 104: 1690-1695)    -   a direct iodosobenzene oxidation (Moriarty et al, Tet.        Lett. (1988) 29: 6913-6916)    -   a two-step conversion via a di-tosyl derivative and an iodo        derivative (DeChristopher et al, JACS (1969) 91:2384-2385).

The following Examples 1 to 9 illustrate the preparation of materialssuitable for use in the invention.

(MA)LDI-TOF mass spectra were obtained using a Voyager EliteBiospectrometry Research Station (PerSeptive Biosystems, Vestec MassSpectrometry Products) in a positive ion mode; FAB spectra were acquiredusing a Varian instrument. Analytical thin-layer chromatography wasperformed on the Kieselgel 60 F₂₅₄ precoated aluminium plates (Merck) oraluminium oxide 60 F₂₅₄ precoated aluminium plates (Merck), spots werevisualized under UV or as specified. Column chromatography was performedon silica gel (Merck Kieselgel 60 0.040-0.063 mm) or aluminium oxide(Aidrich aluminium oxide, activated, neutral, Brockmann I, 150 mesh, 58Å).

Reagents for biological experiments, unless otherwise specified, werefrom Sigma-Aidrich. ¹³C-glucose was from Sigma and Reakhim (Russia).

Reagents obtained from commercial suppliers were used as received. Allsolvents were from Aidrich; trifluoroacetic acid was from Pierce; HPLCgrade solvents were from Chimmed (Russia), and were used without furtherpurification. (S)-2-Amino-5-cyanopentanoic acid was from Genolex(Russia). Deuterium gas was generated by electrolysis by a GC HydrogenSupply Module (output 6 atm; Himelectronika, Moscow, Russia), usingheavy water as a source. Heavy water (²H₂O, D₂O), NaBD₄ and Na¹³CN werefrom Reakhim (Russia) and Gas-Oil JSC (Russia). DMF was freshlydistilled under reduced pressure and stored over 4 Å molecular sievesunder nitrogen. DCM was always used freshly distilled over CaH₂. THF wasdistilled over LiAlH₄.

Example 1—(S)-2-Amino-4-cyano(¹³C)-butyric acid (a Precursor for ¹³C-Argand ¹³C, ²H₂-Arg)

2.19 g (10 mmol) of N-Boc-homo-Serine (Bachern; desiccated overnightover P₂O₅) was dissolved in 10 ml of a mixture ofacetonitrile/dimethylformamide (1:1). Dry Na¹³CN (Gas-Oil JSC, Russia; 1g, 2 eqv) and NaI (10 mg, cat) were added, and the mixture was degassed.Me₃SiCl (2.55 ml, 2 eqv) was then added with a syringe at RT underargon. The reaction mixture was stirred under argon at 60° C. for 6 h,with monitoring by TLC (chloroform/methanol 2:1, visualization in iodinevapor). Upon completion, the reaction mixture was cooled to RT, dilutedwith water (100 ml) and extracted with diethyl ether (2×50 ml). Theorganic phase was washed with water (4×50 ml) and brine (50 ml), dried(Na₂SO₄), decanted and concentrated in vacuo to yield (2.07 g, 91%) ofcolorless oil. The structure of the Boc-nitrile was confirmed byMALDI-TOF (Voyager Elite, PerSeptive Biosystems), with HPA as a matrix.Found: 229.115 (MI), 230.114 (MI+H⁺), 252.104 (MI+Na⁺). No peaks relatedto the starting material were detected.

The removal of the Boc protecting group and the work-up were carried outusing a standard peptide synthesis protocol (50% TFA in DCM, 30 min,RT). The structure of the nitrile was confirmed by MALDI-TOF (VoyagerElite, PerSeptive Biosystems), with with as a matrix. Found: 129.062(MI), 130.070 (MI+H⁺). No signal related to the starting material wasdetected.

Example 2—(S)-2-Amino-4-cyano-butyric acid (a Precursor for 2H2-Arg)

4.93 g (20 mrnol) of N-Boc-L-Glutamine (Sigma) was dissolved in 30 ml ofanhydrous THF and added with stirring to & mixture of triphenylphosphine(10.49 g, 40 mmoi, Aldrich) and 40 ml of anhydrous tetrachloromethane.The reaction mixture was stirred with gentle heating for 3 h (control byTLC, chloroform/methanol 2:1, visualization in iodine vapour), cooledand the precipitate of triphenylphosphine oxide filtered off. The oilobtained upon evaporation and re-evaporation with an additional 15 ml ofTHF was diluted with 30 ml of water. The aqueous fraction was saturatedwith brine, washed with diethyl ether (2×20 mi), and acidified to pH 3.5with sulphuric acid. The product was extracted with ethyl acetate (2×20ml). Combined organic fractions were dried (brine, Na₂SO₄) decanted andevaporated to give 3.46 g (76%) of colorless oil. The structure of theBoc-nitrile was confirmed by MALDI-TOF (Voyager Elite, PerSeptiveBiosystems), with HPA as a matrix. Found: 228.114 (MI), 229.114 (MI+H⁺),251.103 (MI+Na⁺). No peaks related to the starting material weredetected.

The removal of the Boc protecting group and the work-up were carried outusing a standard peptide synthesis protocol (50% TFA in DCM, 30 min,RT). The structure of the nitrile was confirmed by MALDI-TOF (VoyagerElite, PerSeptive Biosystems), with HPA as a matrix. Found: 128.069(MI), 129.075 (MI+H⁺). No signal related to the starting material wasdetected.

Example 3—Lys-²H₂

(S)-2-amino-5-cyanopentanoic acid (Genolex, Russia; 14.21 g, 100 mmol)was dissolved in 100 ml of methanol. To this, Raney nickel, preparedfrom 4 g of alloy (30% Ni) according to (Adkins H, et al, Org,Syntheses. Coll. Vol. Ill, 1955, p. 180) was added, and the reactionmixture was shaken under deuterium (100 atm) at 90° C. for 24 h. (TLC:n-butanol-pyridlne-acetic acid-water: 15-10-3-12: visualization byiodine vapor and fluorescamine). The reaction mixture was filtered andevaporated in vacuo. The product was redissolved in water-ethanol (3:1;20 ml) followed by evaporation in vacuo (×4) and then crystallized fromethylacetate to give 11.55 g (78%) of the deuterated product. Thestructure of deuterated lysine was confirmed by MALDI-TOF (VoyagerElite, PerSeptive Biosystems), with HPA as a matrix. Found: 148.088(MI), 149.089 (MI+H⁺).

Example 4—(5-¹³C, 5,5-²H₂)-Arginine

The (S)-2-Amino-4-cyano(¹³C)-butyric acid (182 mg, 1.41 mmol) andCoCl₂×6H₂O (Aldrich, 670 mg, 2.82 mmol) were dissolved in water (6 ml)and NaBD₄ (ReakMm, Russia; 540 mg. 14.1 mmol) was added in two portionsover 20 min. The nitrile was reduced in 30 min (control by TLC:n-butanol-pyridine-acetic acid-water: 15-10-3-12; fluorescamine/UVdetection for Boc-protected amino acids, iodine vapor visualisation forunprotected amino acids).

The reaction mixture was quenched by acidification (IM HCl) followed byacetone, and purified by ion exchange (Amberlite IR120P (H⁺), Aldrich).The column was washed with water till neutral pH. The product was thenrecovered by washing the column with NH₄GH (0.3M) followed byevaporation. The resulting ornitine-¹³C, ²H₂ (yield: 158 mg, 83%;MALDI-TOF (Voyager Elite. PerSeptive Biosystems), with HPA matrix.Found: 135.071 (MI), 136.068 (MI+H⁺) was dissolved in water and mixedwith an equal volume of 0.5M O-methylisourea (Kimmel, supra), pH 10.5,adjusted with NaOH. After 4-5 h 1% TFA was added to stop the reaction(Bonetto et al. supra). The compound was purified by a RP HPLC (Bufferswere A: 0.1% TFA/H₂O; B: 0.1% TFA/(80% MeCN/20% H₂O)), 0-65% B over 40min to give 140 mg (68%); MALDI-TOF (Voyager Elite, PerSeptiveBiosystems), with HPA matrix; found: 177.402 (MI), 178.655 (MI+H⁺).

Example 5—(5,5-²H₂)-Arginine

The title compound was synthesized using the above protocol, startingfrom (S)-2-amino-4-cyano-butyric acid (Technomm, Russia). MALDI-TOF(Voyager Elite, PerSeptive Biosystems), with HPA matrix; found: 176.377(MI), 177.453 (MI+H⁺).

Example 6—11,11-di-deutero-linoleic acid (18:2)

Linoleic acid (7 g, 25 mmol, Aldrich) was dissolved in 25 ml of carbontetrachloride dried over P₂O₅. N-bromosuccinimide (4,425 g, 25 mmol,desiccated overnight over P₂O₅) and 0.05 g AIBN were added, and thereaction mixture in a flask with a reversed condenser was stirred withgentle heating till the reaction was initiated as manifested by anintense boiling (if the reflux is too intense the heating should bedecreased). When succinimide stopped accumulating on the surface, theheating was continued for another 15 mill (about 1 h in total). Thereaction mixture was cooled to RT and the precipitate filtered off andwashed with CCl₄ (2×5 ml). The combined organic fractions wereevaporated and the 11-Bromolinoleic acid obtained was gradually added toa solution of NaBD₄ (390 mg, 10 mmol) in 30 ml of isopropanol. After anovernight stirring, a diluted solution of HCl was slowly added tillthere was no more deuterium gas produced. Upon a standard workup, themono-deuterated acid was brominated and reduced again to yield a targetdi-deutero derivative (bp 230-231° C./15 mm, 4.4 g, 63%). MALDI-TOF MS:mono-bromo derivative, found: 358.202, 360.191 (doublet, approx 1:1,MI); di-deutero derivative, found: 282.251 (MI).

Example 7

11, 11-D₂-Linoleic acid (18:2) was synthesized by treating linoleic acidwith an eqv of a BuLi-tBuK (Sigma-Aidrich) mix in hexane followed byquenching with D₂O. To improve yields this procedure needs to berepeated 3-4 times. It was found that this procedure also generates adetectable amount of alpha-deuterated product (FAB MS, Xe ions,thioglycerine: found: 283.34 (72; MI+1)⁺, 284.33 (11;alpha-monodeuteroderivative, MI+1)^(÷), 285.34 (10;alpha-dideuteroderivative, MI+1)⁺; the nature of ‘284’ and ‘285’ peakswas established using MS/MS. The substitution at alpha-position can beprevented by utilizing transient ortho-ester protection (Corey & RajuTetrahedron Lett. (1983) 24: 5571), but this step makes the preparationmore expensive.

Example 8—8-D-Deoxyguanosine from Deoxyguanosine

Deoxyguanosine (268 mg, 1 mmol, Aldrich) was dissolved in 4 ml of D₂O.10% Pd/C (27 mg, 10 wt % of the substrate, Aldrich) was added, and themixture was stirred at 160° C. in a sealed tube under B₂ atmosphere for24 h. After cooling to RT, the reaction mixture was filtered using amembrane filter (Millipore Millex®-LG). The filtered catalyst was washedwith boiling water (150 ml), and the combined aqueous fractions wereevaporated in vacuo to give deoxyguanoside-J as a white solid (246 mg,92%). The structure of the nucleoside was confirmed by MALDI-TOF(Voyager Elite, PerSeptive Biosystems), with HPA as a matrix. Found:268.112 (MI).

Example 9—8-D-Deoxyguanosine from 8-bromodeoxyguanosine

7% Pd/C catalyst, prepared from PdCl₂ as described in Chiriac et al(1999) 42: 377-385, was added to a solution of 8-bromodeoxyguanosine(Sigma) and NaOH in water. The mixture was stirred in D₂ (2 atm) at 30°C. The catalyst was filtered off and the reaction mixture wasneutralized with 2N HCL The procedure provides approx. 85-90% yield ofthe product. Other reducing agents can be employed, such as NaBD₄ (seethe synthesis of D,D-linoleic acid).

The following Examples 10 to 12 illustrate the utility of the invention.In order to establish a range of a potential heavy isotope substitutionsfor the invention (from 100% light isotope to 100% heavy isotope, aswell as the localized site protection such as that shown in FIGS. 1-4,using compounds as shown above), and to test for a possible toxicity oflarge amounts of heavy isotopes on an organism, the influence of heavycarbon (¹³C) and specifically “protected’ building blocks of biopolymers(nucleic acid components (nucleosides), lipids and amino acids) on thelife span was tested on a nematode Caenorhabditis elegans.

Previous studies of the model organism C. elegans have almostexclusively employed cultivation on a bacterial diet. Such cultivationintroduces bacterial metabolism as a secondary concern in drug andenvironmental toxicology studies (specific metabolite-deficientbacterial strains can be employed to evaluate the influence ofparticular essential nutrients on the nematode longevity). Axeniccultivation of C. elegans can avoid these problems, yet some earlierwork suggests that axenic growth is unhealthy for C. elegans. (Szewczyket al, Journal of Experimental Biology 209, 4129-4139 (2006)). For thepresent invention, both NGM aad axenic diets were employed incombination with isotopically enriched nutraceutical components.

Example 10

¹³C₆-glucose (99% enrichment; Sigma) was used as a carbon food sourcefor culturing of Escherichia coli; the control was identical except forthe ²C₆-glucose. C. elegans (N2, wild type) were grown on a standard(peptone, salts and cholesterol) media seeded with Escherichia coliprepared as described above. The only carbon-containing component apartfrom E. coli was ¹²C-cholesterol (Sigma; a hormone precursor that isessential for C. elegans), since the corresponding ¹³C-derivative wasunavailable. Nematodes were thus grown on a ‘heavy’ and ‘light’(control) diet in the temperature range of 15-25° C., in pools of 50-100worms each. The animals on both diets developed normally with all majorcharacteristics being very similar.

The longevity data was analyzed using Prism software package (GraphPadsoftware, USA), according to published procedures (Larsen et al,Genetics 139: 1567 (1995)). It was found that animals on the ‘heavy’diet have an increase of a lifespan of around 10% (in a typicalexperiment, 14 days for ¹²C animals versus about 15.5 days for the¹³C-fed worms, for 25° C.).

Example 11

Basic composition of the axenic media used is adapted from (Lu & GoetschNematologica (1993) 39: 303-311). Water-soluble and TEA-solublecomponents (vitamins and growth factors), salts, non-essential aminoacids, nucleic acid substituents, other growth factors and the energysource are prepared as described (0.5 L of 2×). To this, a mix ofessential amino acids is added, containing (for 0.5 L as 2×): 0.98fL-(D₂)-Arg (see above); 0.283 g L-HYS; 1.05 g L-(D₂)-Lys (see below);0.184 g L-Trp; 0.389 g L-Met; 0.717 g L-Thr; 1.439 g L-Leu; 0.861 gL-Ile; 1.02 g L-Val, and 0.623 g L-Phe. Prior to adding to the remainingcomponents, this mixture is stirred at 55° C. for 4 hours until a clearsolution is formed, and then cooled to room temperature.

C. elegans (N2, wild type) are cultivated on this medium. For thecontrol experiment, nematodes are grown on a medium prepared as abovebut containing standard L-Arg and L-Lys instead of the deuteratedanalogues, in the temperature range of 15-25° C., in pools of 50-100worms each. The longevity data is analyzed using Prism software, asdescribed in Example 10.

Example 12

A ¹²C-NGM diet is enriched with 5,5-di-deutero-arginine and6,6-di-deutero-lysine, 11,11-di-deutero-linoleic acid (18:2), and8-D-deoxyguanosine. C. elegans are grown on a standard (peptone, saltsand cholesterol) medium seeded with Escherichia coli prepared asdescribed above, to which deuterium-‘reinforced’ derivatives (see above)are added, to a total concentration of 1 g/L of each deuteratedcompound. Nematodes are thus grown on a ‘heavy’ and ‘light’(control—whereby non-deuterated L-Arginine, L-Lysine, linoleic acid(18:2), and deoxyguanosine are used instead of deuterated analogues in 1g/L concentrations) diet in the temperature range of 15-25° C., in poolsof 50-100 worms each. The longevity data is analyzed using Prismsoftware package, as described in Example 10.

What is claimed is:
 1. A medicament composition, comprising an effectiveamount of an isotopically modified compound, wherein the isotopicallymodified compound reduces oxidative damage of a bodily constituent in apatient after the isotopically modified compound is administered andincorporated into the bodily constituent, wherein the isotopicallymodified compound is a nucleic acid, lipid, or amino acid, and whereinthe lipid is a fatty acid or fatty acid ester isotopically modified atleast at one or more bis-allylic positions.
 2. The medicamentcomposition of claim 1, wherein the isotopically modified compound is alipid.
 3. The medicament composition of claim 2, wherein the lipid is adeuterated fatty acid.
 4. The medicament composition of claim 3, whereinthe deuterated fatty acid is a deuterated ω-3 fatty acid or a deuteratedω-6 fatty acid.
 5. The medicament composition of claim 3, wherein thedeuterated fatty acid is deuterated at least at one or more bis-allylicpositions.
 6. The medicament composition of claim 3, wherein thedeuterated fatty acid is 11-D-linoleic acid or 11,11-D2-linoleic acid.7. The medicament composition of claim 2, wherein the lipid is adeuterated fatty acid ester.
 8. The medicament composition of claim 7,wherein the lipid is a 11-D-linoleic acid ester or 11,11-D2-linoleicacid ester.
 9. The medicament composition of claim 2, wherein the bodilyconstituent of the patient is a fat.
 10. The medicament composition ofclaim 1, wherein the isotopically modified compound is a nucleic acid oran amino acid.
 11. The medicament composition of claim 10, wherein theamino acid is selected from the group consisting of Phe, Val, Trp, Thr,Ile, Met, His, Leu, Lys and Arg.
 12. The medicament composition of claim10, wherein the amino acid is selected from the group consisting ofLys-D₂, Arg-D₂, Trp-D, Thr-D, Phe-D₄, His-D, Lys-D₃ ¹³C₂, Arg-D₃ ¹³C₂,Trp-D₄ ¹³C₄, Val-D₃ ¹³C₃, Thr-D₂ ¹³C₂; (S)-2,6-diaminohexanoicacid-6,6-D₂, (S)-2-amino-5-guanidino pentanoic acid-5-¹³C,5,5-D₂, and(S)-2-amino-5-guanidino pentanoic acid-5,5-D₂.
 13. The medicamentcomposition of claim 1, wherein the medicament composition is in a formof a tablet or pill.
 14. The medicament composition of claim 1, whereinthe medicament composition is in a form suitable for intravenousdelivery.
 15. The medicament composition of claim 1, wherein themedicament composition is in a liquid form.
 16. A method for reducingoxidative damages in vivo in a human, comprising repeatedlyadministering to the human a medicament composition, comprising aneffective amount of an isotopically modified compound, wherein theisotopically modified compound reduces oxidative damage of a bodilyconstitutent in the human after the isotopically modified compound isadministered and incorporated into the bodily constitutent, wherein theisotopically modified compound is a nucleic acid, lipid, or amino acid,and wherein the lipid is a fatty acid or fatty acid ester isotopicallymodified at least at ione or more bis-allylic positions.
 17. The methodof claim 16, wherein the isotopically modified compound is a lipid. 18.The method of claim 17, wherein the lipid is a deuterated fatty acid.19. The method of claim 18, wherein the deuterated fatty acid isdeuterated at least at one or more bis-allylic positions.
 20. The methodof claim 18, wherein the deuterated fatty acid is 11-D-linoleic acid or11,11-D2-linoleic acid.
 21. The method of claim 17, wherein the lipid isa deuterated fatty acid ester.
 22. The method of claim 21, wherein thelipid is a 11-D-linoleic acid ester or 11,11-D2-linoleic acid ester.